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Abstract

Background

DNA interstrand cross-links (ICLs) are critical lesions produced by several cancer
chemotherapy agents including platinum drugs and nitrogen mustards. We have previously
shown in haematological (multiple myeloma) and solid tumours (ovarian cancer) that
clinical sensitivity to such agents can result from a defect in DNA ICL processing
leading to their persistence. Conversely, enhanced repair can result in clinical acquired
resistance following chemotherapy. The repair of ICLs is complex but it is assumed
that the ‘unhooking’ step is common to all ICLs.

Methods

Using a modification of the single cell gel electrophoresis (Comet) assay we measured
the formation and unhooking of melphalan and cisplatin-induced ICLs in cell lines
and clinical samples. DNA damage response in the form of γ-H2AX foci formation and
the formation of RAD51 foci as a marker of homologous recombination were also determined.
Real-time PCR of 84 genes involved in DNA damage signalling pathways was also examined
pre- and post-treatment.

Results

Plasma cells from multiple myeloma patients known to be clinically resistant to melphalan
showed significant unhooking of melphalan-induced ICLs at 48 hours, but did

not

unhook cisplatin-induced ICLs. In ovarian cancer cells obtained from patients following
platinum-based chemotherapy, unhooking of cisplatin-induced ICLs was observed at 48
hours, but no unhooking of melphalan-induced ICLs. In vitro, A549 cells were proficient at unhooking both melphalan and cisplatin-induced ICLs.
γ-H2AX foci formation closely followed the formation of ICLs for both drugs, and rapidly
declined following the peak of formation. RPMI8226 cells unhooked melphalan, but

not

cisplatin-induced ICLs. In these cells, although cross-links form with cisplatin,
the γ-H2AX response is weak. In A549 cells, addition of 3nM gemcitabine resulted in
complete inhibition of cisplatin-induced ICL unhooking but no effect on repair of
melphalan ICLs. The RAD51 foci response was both drug and cell line specific. Real
time PCR studies highlighted differences in the damage response to melphalan and cisplatin
following equi-ICL forming doses.

Conclusions

These data suggest that the mechanisms by which melphalan and cisplatin-induced ICLs
are ‘unhooked’ in vitro are distinct, and the mechanisms of clinical acquired resistance involving repair
of ICLs, are drug specific.

Keywords:

Background

DNA cross-linking agents such as the nitrogen mustards (melphalan, chlorambucil, cyclophosphamide,
ifosfamide), platinum drugs (cisplatin, carboplatin, oxaliplatin), chloroethylnitrosoureas
(carmustine, lomustine), the alkylalkane sulphonate busulphan and the natural product
mitomycin C are widely used drugs as both single agents (including in a high-dose
setting) and as components of many combination chemotherapy regimens
[1,2]. In addition, more selective cross-linking agents such as SJG-136 (SG2000) continue
to be developed
[3,4]. Bi-functional covalent modification (cross-linking) of DNA is essential for antitumor
activity with these drugs
[5,6]. In particular, the DNA interstrand cross-link (ICL) which covalently links together
bases on opposite strands of the DNA helix, and which normally only account for a
small proportion (1-10%) of total DNA adducts, present a serious threat to cellular
survival because they inhibit fundamental processes such as DNA replication and transcription
[1,7]. There is clear evidence that the formation and subsequent persistence of ICLs correlates
with in vitro cytotoxicity
[8,9].

In a previous study, using a modification of the single cell gel electrophoresis (Comet)
assay, we measured formation and repair of DNA ICLs in plasma cells from melphalan-naïve
and melphalan-treated patients (i.e. those who had relapsed after a melphalan-conditioned
autologous stem cell transplant or oral melphalan therapy)
[9]. Similar levels of dose-dependent DNA ICLs were observed in cells from both melphalan-naïve
and treated patients. However, marked differences in ICL repair were observed: cells
from naïve patients had no repair, whereas those from treated patients exhibited between
42-100% repair at 40 hours. In vitro sensitivity to melphalan in plasma cells was found to correlate with ICL repair.
These findings suggest that a defect in ICL repair may contribute to the initial sensitivity
to melphalan and that ICL repair may be an important mechanism by which melphalan
acquired resistance emerges in the clinic
[10,11].

In a second study we examined ICL formation and repair in tumour cells isolated from
fifty ovarian cancer patients
[12]. No significant difference in the peak level of ICL formation in tumour cells was
observed between patients who were either newly diagnosed, or previously treated with,
platinum-based chemotherapy (or between tumour and mesothelial cells from the same
patient). In contrast, the repair of ICLs was much greater in the group of treated
patients. In eight patients it was possible to obtain tumour samples prior to any
chemotherapy, and also at relapse or at interval de-bulking surgery following platinum
chemotherapy. In these patients the mean % repair prior to therapy was 2.85 rising
to 71.23 following treatment. These data again suggest that inefficient repair of
ICLs contributes to the initial clinical sensitivity, and that increased ICL repair
contributes to clinical acquired resistance.

Repair of ICLs is complex and requires the concerted action of multiple pathways
[7,13,14]. Although the exact molecular mechanisms have yet to be fully elucidated, it is clear
that incision around the lesion to allow ‘unhooking’ of the ICL from one of the two
DNA strands represents a pivotal step in the repair process as it relieves the torsional
stress an ICL imposes on the DNA helix and permits processing of the repair intermediates
by downstream pathways. This is the step in ICL repair that can be measured using
the modification of the comet assay since it detects the ability of the DNA strands
to separate under alkaline conditions. A number of nucleases have been suggested to
play such a role in this unhooking step, including the XPF-ERCC1 complex
[5,15,16] and the Fanconi anaemia pathway orchestrates incisions at sites of crosslinked DNA
(recently reviewed in
[17]. Regardless of the exact mechanism of unhooking, it is widely assumed that this unhooking
step will be common to all DNA ICLs. In this study, however, we present evidence in vitro and in clinical samples with acquired resistance that the mechanisms of unhooking
for melphalan and cisplatin-induced ICLs are distinct.

Methods

Cell lines and peripheral blood lymphocytes

A549 and RPMI8226 cell lines were purchased from the European Collection of Cell Cultures
(ECACC). The human ovarian cancer cell line A2780 was established from tumour tissue
from an untreated patient
[18]. Growing A2780 cells in cisplatin and selecting for cisplatin resistance generated
the stably resistant A2780cisR cell line. Both cell lines were obtained from Dr Swee
Sharp, Institute of Cancer Research, Sutton, UK. RPMI8226 cells was maintained in
RPMI1640 media containing 2 mM L-glutamine and 10% foetal calf serum (FCS). A549 was
maintained in Dulbecco’s Modified Eagles Medium (DMEM) containing 2 mM L-glutamine
and 10% FCS. All cell lines were maintained in a humidified atmosphere at 37°C with
5% carbon dioxide (CO2) and maintained in exponential growth. The cells were kept at low passage, returning
to original frozen stocks every 3 to 6 months, and tested regularly for Mycoplasma.

Peripheral blood lymphocytes (PBLs) were isolated using the Vacutainer® CPT™ system
(Becton Dickinson, Oxford, UK). Samples were centrifuged at 1500 g for 20 minutes
at room temperature. The fluffy mononuclear layer at the interface of the two layers
was removed using a Pasteur pipette and transferred to a 15 ml tube. 10 ml cold RPMI
1640 tissue culture media was then added and the tube gently inverted and centrifuged
immediately at 200 g for 5 minutes at 4°C. The supernatant was then discarded and
the cell pellet re-suspended in RPMI 1640 containing 10% foetal calf serum and 2 mM
L-glutamine.

Patient samples

Plasma cells were isolated from bone marrow taken from Multiple Myeloma patients using
standard Ficoll-Hypaque
[10]. Patients 1 and 2 had relapsed following vincristine, adriamycin and dexamethasome
(VAD) chemotherapy, received a melphalan conditioned (200 mg/m2) autologous stem cell transplant and were known to be clinically melphalan resistant.
All samples studied contained in excess of 80% plasma cells.

Ovarian cancer tumour cells taken from ovarian cancer patients either pre- or post-platinum
based chemotherapy, were isolated from ascitic fluid as described in detail elsewhere
[12]. Ethics approval was gained from the Joint UCL/UCLH Committee on the Ethics of Human
Research. In order to separate tumour cells from non-tumour mesothelial cells, ascitic
fluid was centrifuged at 200 g for 5 minutes. Cell pellets were re-suspended in DMEM
containing 10% FCS and 2 mM L-glutamine and seeded into large tissue culture flasks
and incubated in a humidified atmosphere at 37°C with 5% CO2. After 1 hour, the entire volume of tissue culture medium in each flask containing
unattached cells was transferred into a fresh tissue culture flask and DMEM containing
10% FCS and 2 mM L-glutamine was replaced in the original flasks. Normal mesothelial
cells attached to the plastic surface within the first hour, where as tumour cells
required a longer period of time to detach in response to trypsin. Further purification
of the tumour samples were achieved using trypsinisation until the contaminant mesothelial
cells were seen to detach, while the tumour cells remained in situ.

Drug treatment

Cell lines, PBLs and patient samples were incubated with either melphalan (Sigma Chemical
Co., Poole, U.K.) or cisplatin (David Bull Laboratories, Australia) for 1 hour at
37°C and 5% CO2 in a humidified atmosphere. For RPMI8226 cell line, PBLs and myeloma plasma cells,
the drug was removed by centrifugation at 200 g for 5 minutes, the supernatant removed
and cells re-suspended in drug-free full media. Cells were then incubated at 37°C
and 5% CO2 in a humidified atmosphere. For A549, A2780, A2780cisR cell lines and patient ovarian
tumour cells isolated from ascitic fluid, drug treatments were carried out in 6-well
plates and the media replaced with drug free medium following treatment. In order
to assess DNA interstrand cross-linking and repair, samples were taken at various
time points following the 1 hour drug incubation. For combination experiments, cells
were treated with 3nM gemcitabine (Eli Lilly & Company, Basingstoke, U.K.) in combination
with melphalan or cisplatin. Both drugs were removed as described above and cells
incubated with 3nM gemcitabine for the remainder of the incubation period.

Determination of DNA interstrand cross-link formation and its repair using the single
cell gel electrophoresis (comet) assay

The details of the single cell gel electrophoresis (comet) assay used to measure DNA
interstrand cross-linking and repair are described in detail elsewhere
[19]. All procedures were carried out on ice and in subdued lighting. All chemicals were
obtained from Sigma Chemical Co. (Poole, U.K.) unless otherwise stated. Immediately
before analysis cells were diluted to give a final concentration of 2.5 x104 cells/mL and irradiated (15 Gy) in order to deliver a fixed number of random DNA
strand breaks. After embedding cells in 1% agarose on a pre-coated microscope slide,
the cells were lysed for 1 hour in lysis buffer (100 mM disodium EDTA, 2.5 M NaCl,
10 mM Tris–HCl pH 10.5) containing 1% Triton X-100 added immediately before analysis,
and then washed every 15 minutes in distilled water for 1 hour. Slides were then incubated
in alkali buffer (50 mM NaOH, 1 mM disodium EDTA, pH12.5) for 45 minutes followed
by electrophoresis in the same buffer for 25 minutes at 18 V (0.6 V/cm), 250 mA. The
slides were finally rinsed in neutralising buffer (0.5 M Tris–HCl, pH 7.5) then saline.

After drying the slides were stained with propidium iodide (2.5 μg/mL) for 30 minutes
then rinsed in distilled water. Images were visualised using a NIKON inverted microscope
with high-pressure mercury light source, 510-560 nm excitation filter and 590 nm barrier
filter at x20 magnification. Images were captured using an on-line CCD camera and
analysed using Komet Analysis software 4.02 (Andor Technology, U.K.). For each duplicate
slide 25 cells were analysed. The tail moment for each image was calculated as the
product of the percentage DNA in the comet tail and the distance between the means
of the head and tail distributions
[20]. DNA interstrand cross-linking was expressed as percentage decrease in tail moment
compared to irradiated controls calculated by the formula:

In cells treated with DNA cross-linking agents and gemcitabine in combination, cross-linking
was expressed as percentage decrease in tail moment compared to irradiated controls
calculated by the formula below. This formula was used to compensate for the additional
single strand breaks induced by gemcitabine in addition to those produced by the irradiation
step.

In both multiple myeloma and ovarian patient samples, percentage repair was calculated
at 48 hours following the peak of DNA interstrand cross-linking (9 hours for cisplatin
[12] and 16 hours for melphalan
[10]).

Measurement of γ-H2AX and RAD51 foci by immunofluorescence

For the A549 cell line, 8 x 104 cells per well were seeded in a 2 well LAB-TEK® II chamber slides™ (Nalgene Nunc
International, Hereford, UK) and incubated overnight at 37°C. Cells were treated with
either 2 μM melphalan or 5 μM cisplatin for 1 hour after which the drug was removed
and cells incubated at 37°C in drug-free medium.

For the RPMI8226 cell line, cells were treated with either 2 μM melphalan or 5 μM
cisplatin for 1 hour after which the drug was removed by centrifugation at 200 g,
cells re-suspended and incubated at 37°C in drug-free medium. At the required time
point, 10 x104 cells were adhered to Vision BioSystems™ Plus slides by cytospinning at 650 rpm for
5 minutes at room temperature. Slides were then dried at room temperature.

For both cell lines, cells were fixed with ice cold methanol: acetone (50:50) for
15 min at 4°C. Cells were washed 3 times with cold PBS then permeabilized with 0.5%
Triton X-100 in PBS for 15 min at room temperature. Cells were then blocked overnight
at 4°C with blocking buffer (0.1% Triton X-100, 0.2% skimmed dry milk in PBS). Blocked
cells were incubated overnight at 4°C with either anti-phospho-histone H2A.X (Ser139)
monoclonal antibody (Millipore, U.K) at a 1:1000 dilution or anti-RAD51 (H-92) polyclonal
antibody (Santa Cruz Biotechnology Inc) at a 1:100 dilution in blocking buffer. After
washing 3 times with wash buffer (0.1% Triton X-100 in PBS), cells were then incubated
for 4 hours at room temperature with Alexa Fluor® 488 goat anti-mouse secondary antibody
(InVitrogen, UK) for γH2AX staining or Alexa Fluor® 488 goat anti-rabbit secondary
antibody (InVitrogen, UK) for RAD51 staining, at a dilution of 1:1000 and 1:200 respectively
in blocking buffer. Cells were then washed with PBS. For γ-H2AX, cells were counterstained
with 2 μg/mL propidium iodide for 2 min. Slides were then rinsed in distilled water
for 30 minutes, mounted with Vectashield® (Vector Laboratories, Peterborough, UK)
and the edges sealed with clear nail polish. For RAD51, slides were mounted with Vectashield®
with DAPI and the edges sealed with clear nail varnish. Images were visualised using
Perkin Elmer Ultraview ERS Suite v 3.0.0 and confocal microscopy consisting of Zeiss
Axiovert 200 inverted fluorescence microscope (x40 oil objective) equipped with 14
bit ECCD camera and argon and krypton gas excitation lasers at 488 nm and 568 nm.
Foci were counted in 50 cells per time point and results are expressed as mean number
of foci per cell from three independent experiments.

Real-time PCR of genes involved in DNA damage signalling pathways

Exponentially growing cells were treated for 1 hour with either 150 μM cisplatin or
50 μM melphalan after which the drug was removed and replaced with drug free media.
Cells were then incubated for 9 hours (cisplatin) and 16 hours (melphalan) post-treatment
to allow maximum formation of interstrand cross-links. Cells were then trypsinised,
washed with PBS and pelleted and stored at -80°C prior to analysis.

Total RNA was extracted from the cell pellets using a RNEasy kit (Qiagen) according
to the manufacturers protocol and concentration measured. Template cDNA was generated
from 1 μg of RNA using the RT2 First Strand Kit (SABiosciences-Qiagen). This template cDNA was then amplified in
25 μl volumes using the DNA Damage Signalling Pathway PCR Array and RT2 qPCR Mastermix (SABiosciences-Qiagen). Amplification was carried out in an Applied
Biosystems 7500 RT-PCR machine. The RT-PCR condition was an initial incubation at
95°C for 10 minutes followed by 45 cycles at 95°C (15 seconds) and 60°C (1 minute).

The Functional Gene Groupings and Gene Table are shown in Supplementary Material.

Results and discussion

Different mechanisms of ‘unhooking’ of DNA ICLs in clinical acquired resistant tumour
samples

We have previously shown that plasma cells from myeloma patients prior to any chemotherapy
treatment are defective in ‘unhooking’ melphalan-induced ICLs when treated ex vivo, whereas cells from patients following treatment who become clinically resistant
to melphalan are proficient in unhooking melphalan ICLs
[10]. We examined the ability of plasma cells from two representative melphalan resistant
patients to unhook the ICLs produced by cisplatin, in addition to melphalan (Figure
1A). Cells were treated ex vivo with either melphalan (50 μM) or cisplatin (150 μM) for 1 hour, drug removed and
the level of ICLs measured with time using the established modification of the single
cell gel electrophoresis (comet) assay
[19]. The decrease in level of ICLs at 48 hour (expressed as the % repair at 48 hours
in Figure
1A) was compared to the 16 hour (melphalan) and 9 hour (cisplatin) levels, which we
have previously shown to be the time of peak ICL for these agents
[10,12]. In both these patient samples a significant repair (unhooking) of melphalan-induced
ICLs was observed (40% in patient 1 and 58% in patient 2), as has been observed previously
[10]. In contrast, no unhooking of cisplatin-induced ICLs was observed at 48 hours in
either patient sample. In fact, in the cells from patient 2, the level of ICLs at
48 hours was slightly higher than at 9 hours resulting in the negative ‘repair’ value.

Figure 1.Unhooking of DNA ICLsproduced by melphalan orcisplatin in clinical samplesmeasured using the singlecell gel electrophoresis (comet)assay.A: Plasma cells from two multiple myeloma patients clinically resistant to melphalan.
Cells were treated ex vivo for 1 hour with either 150 μM cisplatin or 50 μM melphalan after which the drug was
removed and replaced with drug free media. Cells were then incubated for 9 hours (cisplatin,
blue) or 16 hours (melphalan, red) post-treatment to allow maximum formation of interstrand
cross-links. The level of ICL at this time point was compared to a second sample incubated
for a total of 48 hours to obtain the % repair. B: As above but in tumour samples from ovarian cancer patients. In this case the two
patients provided samples on initial diagnosis and prior to any platinum-based chemotherapy
(Pre) and again following relapse on platinum-based therapy (Post).

In a further two patient samples that showed 30% and 14% unhooking of melphalan-induced
ICLs, no unhooking of cisplatin-induced ICLs was observed. Cells from a melphalan
naïve patient showed no repair of ICLs produced by either drug.

The ability of cancer cells taken from two ovarian cancer patients to unhook the ICLs
produced by the two drugs was then examined. In these patients it was possible to
obtain tumour samples at initial diagnosis (before any chemotherapy) and then again
after the patients had undergone platinum-based chemotherapy. Under the identical
drug-treatment conditions used in Figure
1A, the initial cells (Pre) from neither patient were able to unhook the ICLs produced
by melphalan or cisplatin up to 48 hours (Figure
1B). In contrast, the tumour cells from both patients taken after platinum-based chemotherapy
(Post) showed efficient unhooking of cisplatin ICLs (77% and 60% at 48 hours) but
no unhooking of melphalan ICLs.

In total, 12 pre-chemotherapy and 7 post platinum-based chemotherapy patient samples
were tested for unhooking of melphalan ICLs. The mean % repair (unhooking) was 4%
and 3% for the pre- and post-chemotherapy patients, respectively. This is in marked
contrast to our previously reported data where mean% repair of cisplatin ICLs was
3% pre-chemotherapy and 71% post platinum-based chemotherapy
[12]. Taken together, these data suggest that distinct mechanisms are evoked in the two
tumour types in patients following chemotherapy, resulting in different mechanisms
of unhooking for melphalan and cisplatin-induced ICLs.

Differences in unhooking of melphalan and cisplatin-induced ICLs in human tumour cell
lines

We next looked for cell line models that could replicate the phenotype that we observed
in the clinical samples. The time course of ICL formation and repair was examined
in the human non-small cell lung cancer cell line A549 following a 1 hour treatment
with 50 μM melphalan or 150 μM cisplatin. These drug doses, (which are within the
range of the GI50 values as shown in
1) were chosen to be consistent with our data in clinical samples and to give equivalent
peak levels of ICL by the two agents. Representative comet images are shown in Figure
2A. In A549 cells, the peak of ICL was at 9 hours for cisplatin and 16 hours for melphalan,
and in these cells the ICLs produced by both agents were efficiently unhooked, resulting
in 92% and 81% repair at 48 hours, respectively (Figure
2B). In addition to measuring ICLs using the comet assay, DNA damage response in the
form of γ-H2AX foci formation was also followed in the same cells (Figure
2C,D). Previous studies from our laboratory have shown that γ-H2AX foci formation can
be used as a pharmacodynamic indicator of ICL formation for both nitrogen mustard
and platinum-based drugs
[21]. γ-H2AX is likely marking sites of double strand breaks generated after unhooking
or lesion processing by structure specific endonucleases. Doses of drug used to treat
cells for 1 hour were lower than those used in the comet assay due to the increased
sensitivity of this assay. γ-H2AX foci formation followed the timing of ICL formation
for both drugs, as shown previously in a different cell line
[21], and rapidly declined following the peak of formation (Figure
2D). The decline in γ-H2AX suggests the resolution of the intermediate double strand
breaks by downstream pathways e.g. homologous recombination repair, translesion DNA
synthesis etc. We previously showed that γ-H2AX foci resulting from nitrogen mustard
and cisplatin-induced ICLs persisted longer in homologous recombination defective
cells
[21].

Additional file 1.Table S1. GI50 values (dose of drug that inhibits growth by 50%) for melphalan and cisplatin in
the human A548, RPMI8226 cell lines. Drug exposure was for 1 hour at 37°C and cells
were incubated in drug-free medium for a further 4 days prior to analysis using the
sulforhodamine B assay. Values are mean±s.d. from at least three independent experiments.

Figure 2.DNA ICL and γ-H2AXfoci formation in A549cells following treatment witheither cisplatin or melphalan.A: Representative comet images from A549 cells either untreated, or treated with 50
μM melphalan for 1 hour. Drug-treated samples shown were following a subsequent 16
hours incubation in drug free medium. B: Cells were treated for 1 hour with either 150 μM cisplatin (blue) or 50 μM melphalan
(red) after which the drug was removed and replaced with drug free media. Samples
were taken at different times of post-incubation and ICLs measured using the comet
assay. Data are the mean ± s.d. from at least three independent experiments. C: Representative A549 cells showing γ-H2AX foci following treatment with cisplatin
at 5 μM followed by post-incubation in drug free medium for the times shown. D: Cells were treated with either 2 μM melphalan (red) or 5 μM cisplatin (blue) for
1 hour after which the drug was removed and cells incubated at 37°C in drug-free medium.
Samples were taken at different times of post-incubation and γ-H2AX foci formation
determined. Data are the mean ± s.d. from at least three independent experiments.

Identical experiments were performed in the human myeloma cell line RPMI8226 (Figure
3). In this cell line the peak of melphalan-induced ICLs was again at 16 hours and
significant unhooking was observed within 8 hours (Figure
3A). In contrast, cisplatin ICLs formed by 9 hours but were not unhooked over a 48
hour period. This cell line, therefore, was consistent with the phenotype seen in
the melphalan-resistant plasma cells from patients (Figure
1A). This same phenotype was also observed in a second myeloma cell line U266 (data
not shown). The γ-H2AX foci response in RPMI8226 cells is shown in Figure
3B. Although the response was weaker than that seen in A549 cells, γ-H2AX foci showed
a similar response to melphalan, peaking with the formation of ICLs and then declining
rapidly. With cisplatin, however, although cross-links form, the γ-H2AX response is
extremely weak (Figure
3B). This lack of a significant DNA damage response is consistent with the lack of
unhooking of the ICLs observed in this cell line, therefore preventing the subsequent
generation of double strand breaks.

Figure 3.DNA ICL and γ-H2AXfoci formation in RPMI8226cells following treatment witheither cisplatin or melphalan.A: Cells were treated for 1 hour with either 150 μM cisplatin (blue) or 50 μM melphalan
(red) after which the drug was removed and replaced with drug free media. Samples
were taken at different times of post-incubation and ICLs measured using the comet
assay. Data are the mean ± s.d. from at least three independent experiments. B: Cells were treated with either 2 μM melphalan (red) or 5 μM cisplatin (blue) for
1 hour after which the drug was removed and cells incubated at 37°C in drug-free medium.
Samples were taken at different times of post-incubation and γ-H2AX foci formation
determined. Data are the mean ± s.d. from at least three independent experiments.

We then looked in human ovarian cancer cell lines for a phenotype that would mirror
that observed in the clinical situation shown in Figure
1B. A2780 cells gave peak of cross-linking at 9 hours and 16 hours for cisplatin and
melphalan, respectively, as seen in the other cell lines. These cells were not efficient
at unhooking either type of cross-link giving 0% and 16% repair at 48 hours for cisplatin
and melphalan, respectively (data not shown). This cell line therefore mirrored the
clinical phenotype in chemotherapy naïve ovarian cancer (Figure
1B). A cisplatin acquired resistant line (A2780cisR) derived from A2780 was also examined.
In this line the levels of ICLs produced by cisplatin and melphalan were identical
to those in A2780 indicating that the mechanism of drug resistance could not be attributed
to an altered transport mechanism or intracellular detoxification of the drug. This
is in contract to other reports in the literature e.g. Jansen et al. 2002
[22] in which A2780cisR cells are shown to have elevated glutathione. The A2780cisR cell
line used in the present study differed from the parental line, however, in that it
was now capable of unhooking the cross-links produced by both agents. Since these
cells efficiently unhook both types of cross-link, it is not representative of the phenotype observed clinically
where only cisplatin-induced ICLs were repaired (Figure
1B).

p53 is one of the most important factors in determining the sensitivity of cells to
DNA damage. The A549 and A2780 cell lines are both p53 wild type
[23] and the RPMI8226 cell line p53 mutant
[24]. Since A549 cells unhook the ICLs produced by both melphalan and cisplatin, RPMI8226
cells unhook only melphalan ICLs and A2780 unhook neither, p53 status does not explain
these findings.

Effect of gemcitabine on the unhooking of cisplatin and melphalan-induced ICLs

Gemcitabine has previously been shown to act synergistically with cisplatin in vitro[25] and the combination with platinum drugs is useful clinically
[26-28]. We examined the effect of continuous administration of 3nM gemcitabine on the repair
(unhooking) of cisplatin and melphalan-induced ICLs in A549 cells (Figure
4). In the case of cisplatin, gemcitabine completely inhibited the unhooking of ICLs
with 0% repair at 48 hours compared to 85% in the absence of gemcitabine (Figure
4A). We have observed a similar inhibition of repair in lymphocytes from patients treated
with the combination of carboplatin and gemcitabine
[28] and fludarabine has been shown to suppress DNA ICL removal in chronic lymphocytic
leukemia lymphocytes
[29]. In contrast, gemcitabine at 3nM had no effect on the removal of melphalan-induced ICLs in A549 cells (Figure
4B). This again suggests that the mechanisms of unhooking for cisplatin and melphalan
ICLs are distinct, with only the former mechanism being inhibited by gemcitabine.
The mechanism by which gemcitabine inhibits the unhooking of cisplatin ICLs remains
unclear. Gemcitabine is believed to inhibit nucleotide excision repair by incorporation
into repair patches thereby causing chain termination. One possible mechanism is that
the nucleotide excision repair of cisplatin-induced intrastrand adducts is inhibited by incorporation of gemcitabine into repair patches resulting
in sequestering of repair proteins, including those required for the initial unhooking
step of DNA ICLs.

Figure 4.Effect of gemcitabine onthe unhooking of cisplatinor melphalan-induced ICLs inA549 cells.A: Cells were treated for 1 hour with 150 μM cisplatin either alone (blue) or in the
presence of 3nM gemcitabine (green) after which the drugs were removed and replaced
with drug free media, or media containing 3nM gemcitabine. Samples were taken at different
times of post-incubation and ICLs measured using the comet assay. Data are the mean
± s.d. from at least three independent experiments. B: Cells were treated for 1 hour with 50 μM melphalan either alone (red) or in the
presence of 3nM gemcitabine (green) after which the drugs were removed and replaced
with drug free media, or media containing 3 nM gemcitabine. Samples were taken at
different times of post-incubation and ICLs measured using the comet assay. Data are
the mean ± s.d. from at least three independent experiments.

The roles of homologous recombination and replication

The formation of RAD51 foci as a marker of homologous recombination in A549 and RPMI8226
cells was examined following treatment with cross-linking agent (Figure
5). Representative RAD51 images are shown in Figure
5A. In A549 cells, a strong RAD51 foci response followed the peak of ICL for melphalan
and then declined rapidly (Figure
5B), similar to the γ-H2AX response to this drug in this cell line (Figure
2D). The response following cisplatin was, however, distinct in that there was an initial
peak at 4 hours with levels decreasing to baseline at 8 hours followed by a second
late peak at 24 hours (Figure
5B). We have observed this biphasic response to cisplatin in other cell types including
human leukaemic K562 cells and lymphocytes (data not shown). In RPMI8226 cells the
RAD51 response was weak (Figure
5C), despite the formation of cross-links by both agents. Homologous recombination
activity has been shown to be elevated in multiple myeloma cells leading to an increased
rate of mutation and progressive accumulation of genetic variation over time
[30]. Interestingly, the basal expression levels of RAD51 mRNA were 5-fold higher in RPMI8226
cells than in A549 as determined by real-time PCR (data not shown). The lack of a
significant RAD51 foci response to ICLs in RPMI8226 cells is therefore not due to
a lack of RAD51 protein.

Figure 5.The roles of homologousrecombination and replication inthe repair of melphalanand cisplatin-induced ICLs.A: Representative cells showing RAD51 foci following treatment with 5 μM cisplatin
followed by post-incubation in drug free medium for the times shown. B: A549 cells were treated with either 2 μM melphalan (red) or 5 μM cisplatin (blue)
for 1 hour after which the drug was removed and cells incubated at 37°C in drug-free
medium. Samples were taken at different times of post-incubation and RAD51 foci formation
determined. Data are the mean ± s.d. from at least three independent experiments.
C: RPMI8226 cells were treated with either 2 μM melphalan (red) or 5 μM cisplatin (blue)
for 1 hour after which the drug was removed and cells incubated at 37°C in drug-free
medium. Samples were taken at different times of post-incubation and RAD51 foci formation
determined. Data are the mean ± s.d. from at least three independent experiments.
D: Isolated human peripheral blood lymphocytes were treated for 1 hour with either
150 μM cisplatin (blue) or 50 μM melphalan (red) after which the drug was removed
and replaced with drug free media. Samples were taken at different times of post-incubation
and ICLs measured using the comet assay. Data are the mean from at least two independent
experiments.

It might be expected that γ-H2AX and RAD51 foci would form coincidentally. Both A549
and RPMI8226 cells can unhook melphalan-induced ICLs. In A549 cells there is a strong
γ-H2AX response, peaking with the formation of ICLs and then declining rapidly. The
RAD51 response follows the same time course. In contrast, in RPMI8226 cells the γ-H2AX
response to melphalan ICLs is less than half that observed in A549 cells for the equivalent
peak level of ICLs and in RPMI8226 cells there is no significant RAD51 response. The
reasons for the different responses are unclear, however the rapid decrease in γ-H2AX
foci in the RPMI8226 cells in the absence of RAD51 foci suggests that resolution of
double strand breaks may not be by homologous recombination repair in these cells.

The processing of ICLs may differ in replicating and non-replicating cells
[31]. We examined the ability of isolated non-replicating human lymphocytes to unhook
cisplatin and melphalan-induced ICLs (Figure
5D). The peak of cross-linking for both drugs was as observed in the human tumour cell
lines, and lymphocytes rapidly unhooked the cross-links produced by both drugs. Clearly,
melphalan and cisplatin-induced ICLs can be unhooked in both replicating and non-replicating
cells. Whether the mechanisms involved are the same is unknown and warrants further
investigation.

Expression of DNA damage response genes

In order to look for possible factors which could explain the different repair response
we next examined by real time PCR the effect on expression of 84 genes involved in
DNA damage signalling/repair pathways following exposure of cells to ICL agent. Comparisons
were made at doses and times which gave an equivalent peak of ICL (cisplatin: 150
μM, 1 hour followed by 9 hours post-incubation; melphalan 50 μM, 1 hour followed by
16 hours). Figure
6 shows the results for A549 cells following melphalan (A) or cisplatin (B) treatment.
In each case the mean expression from three individual drug treatment repeat samples
are compared to three control samples. Genes which show increased expression by more
than 2-fold compared to control cells following drug treatment are shown in red and
those which show decreased expression in green. The genes that show more than 2-fold
altered expression in A549 cells and RPMI8226 cells are detailed in Table
1. In A549 cells only five genes (BTG2, HUS1, LIG1, SESN1 and TREX1) show a greater
than 2-fold increased expression following melphalan (Figure
6A, Table
1). In this cell line the same five genes exhibit increased expression after cisplatin
treatment (Figure
6B, Table
1) but, in addition, four other genes (GAD45A, PCBP4, PCNA, XPC) showed increased expression.
The two genes showing the greatest level of increased expression (SESN1 and BTG2)
were the same for the two drugs. The number of genes that are decreased in expression
by more than 2-fold (green) was much greater for cisplatin (13) compared to melphalan
(1).

Figure 6.Real time PCR analysisof the changes inexpression of 84 genesinvolved in DNA damagesignalling/repair pathways, following exposureof cells to ICLagent.A: Prior to RNA extraction, A549 cells were treated with melphalan at 50 μM for 1 hour
followed by 16 hours post-incubation to allow peak ICL formation. The mean expression
from three individual drug treatment repeat samples are compared to three individual
non-drug treated control samples. Genes which show increased expression by more than
2-fold compared to control cells following drug treatment are shown in red and those
which show decreased expression in green. B: As above, but A549 cells were treated with cisplatin at 150 μM for 1 hour followed
by 9 hours post-incubation to allow peak ICL formation.

Table 1.Summary of the genes from A549 or RPMI8226 cells whose expression is changed more
than two-fold following peak ICL formation by ether melphalan or cisplatin treatment
compared to untreated controls

In RPMI8226 cells the pattern of altered expression is distinct from A549 cells (Table
1). For melphalan, a different five genes had increased expression (PCNA, GTSE1, PRKDC,
ZAK, RPL13A), whereas for cisplatin six genes showed increased expression (PCNA, GTSE1,
RPL13A, EXO1, FANCG, FEN1). In this cell line, neither SESN1 nor BTG2 was increased
by either drug. Interestingly, levels of expression of the DNA repair protein ERCC1
did not change by more than two-fold in either cell line following either cross-linking
agent, despite this protein having a potential role in the unhooking step
[5,15,16]. Cells defective in this protein show extreme sensitivity to both nitrogen mustard
and platinum-based drugs
[5,32]. Although the real time PCR data in the current study highlight differences in the
damage response to melphalan and cisplatin in the two cell lines, no clear pattern
emerges which could explain the different response of the cells to unhooking of cisplatin
and melphalan ICLs.

Conclusions

Overall, these data provide conclusive evidence that the mechanisms by which melphalan
and cisplatin-induced ICLs are ‘unhooked’ in vitro are distinct. Only the latter mechanism is inhibited by gemcitabine. Importantly,
the observed mechanisms of clinical acquired drug resistance in multiple myeloma to
melphalan and in ovarian cancer to cisplatin, which involve repair/unhooking of ICLs,
are shown to be specific to the individual drug. This clearly has important clinical
implications for the treatment of drug-resistant disease.

Abbreviation

ICL: Interstrand Cross-link.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

VJS, HL, CN, and KK performed the comet assays, VJS and AB performed the γ-H2AX and
RAD51 assays and JPB the real-time PCR. CC, JAL and CN provided the clinical myeloma
and ovarian cancer samples. JAH conceived the study and drafted the manuscript. JAH
and DH designed and directed the studies. All authors read and approved the final
manuscript.